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Key Points Inhibition of RAB protein function mediates the anti–acute myeloid leukemia activity of statins.Statin sensitivity is associated with enhanced vesicle-mediated traffic., Visual Abstract, Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.

Summary Background Benefits of cord blood transplantation include low rates of relapse and chronic graft-versus-host disease (GVHD). However, the use of cord blood is rapidly declining because of the high incidence of infections, severe acute GVHD, and transplant-related mortality. UM171, a haematopoietic stem cell self-renewal agonist, has been shown to expand cord blood stem cells and enhance multilineage blood cell reconstitution in mice. We aimed to investigate the safety and feasibility of single UM171-expanded cord blood transplantation in patients with haematological malignancies who do not have a suitable HLA-matched donor. Methods This single-arm, open-label, phase 1–2 safety and feasibility study was done at two hospitals in Canada. The study had two parts. In part 1, patients received two cord blood units (one expanded with UM171 and one unmanipulated cord blood) until UM171-expanded cord blood demonstrated engraftment. Once engraftment was documented we initiated part 2, reported here, in which patients received a single UM171-expanded cord blood unit with a dose de-escalation design to determine the minimal cord blood unit cell dose that achieved prompt engraftment. Eligible patients were aged 3–64 years, weighed 12 kg or more, had a haematological malignancy with an indication for allogeneic hematopoietic stem cell transplant and did not have a suitable HLA-matched donor, and a had a Karnofsky performance status score of 70% or more. Five clinical sites were planned to participate in the study; however, only two study sites opened, both of which only treated adult patients, thus no paediatric patients (aged ClinicalTrials.gov , NCT02668315 . Findings Between Feb 17, 2016, and Nov 11, 2018, we enrolled 27 patients, four of whom received two cord blood units for safety purposes in part 1 of the study. 23 patients were subsequently enrolled in part 2 to receive a single UM171-expanded cord blood transplant and 22 patients received a single UM171-expanded cord blood transplantation. At data cutoff (Dec 31, 2018), median follow-up was 18 months (IQR 12–22). The minimal cord blood unit cell dose at thaw that achieved prompt engraftment as a single cord transplant after UM171 expansion was 0·52 × 105 CD34-positive cells. We successfully expanded 26 (96%) of 27 cord blood units with UM171. Among the 22 patients who received single UM171-expanded cord blood transplantation, median time to engraftment of 100 neutrophils per μL was 9·5 days (IQR 8–12), median time to engraftment of 500 neutrophils per μL was 18 days (12·5–20·0), and no graft failure occurred. Median time to platelet recovery was 42 days (IQR 35–47). The most common non-haematological adverse events were grade 3 febrile neutropenia (16 [73%] of 22 patients) and bacteraemia (nine [41%]). No unexpected adverse events were observed. One (5%) of 22 patients died due to treatment-related diffuse alveolar haemorrhage. Interpretation Our preliminary findings suggest that UM171 cord blood stem cell expansion is feasible, safe, and allows for the use of small single cords without compromising engraftment. UM171-expanded cord blood might have the potential to overcome the disadvantages of other cord blood transplants while maintaining the benefits of low risk of chronic GVHD and relapse, and warrants further investigation in randomised trials. Funding Canadian Institutes of Health Research, Canadian Cancer Society and Stem Cell Network.

Nanomedicines, including liposomes, have been used to improve the clinical efficacy and safety of drugs. In some liposomal formulations, a hydrophilic polymer coating of poly(ethylene glycol) (PEG) is used to increase the circulation time. Understanding the biological mechanisms responsible for the clearance of PEGylated and non-PEGylated nanomedicines is necessary to develop better-performing materials. The purpose of this work is to explore the role of complement in the elimination of intravenously administered liposomes (PEGylated and non-PEGylated) in mice and rats. Here, the complement cascade was depleted by intraperitoneal injections of cobra venom factor (CVF) 12 and 24 hours before the intravenous injection of radiolabeled liposomes. In both mice and rats, non-PEGylated liposomes showed faster elimination than PEGylated liposomes. At a lipid dose of 20 mg kg−1, the abrogation of the complement cascade (in CVF group) did not alter the circulation time of either PEGylated or non-PEGylated liposomes. In contrast, at lower doses (2 mg kg−1), animals treated with CVF had slightly higher levels of circulating liposomes, especially during the 24 hours pharmacokinetic studies. The complement cascade seems to govern the uptake of non-PEGylated liposomes by splenic B cells. Altogether, these results suggest that although PEGylated and non-PEGylated liposomes can activate complement, the impact of this cascade on their circulation time is minor and mostly perceivable at later phases of distribution. This work enlightens biological pathways responsible for in vivo clearance of liposomes and will help in orienting future research in elucidating the nano-bio interface.

Simple Summary The immune system is the first line of protection against infected and tumor cells. Macrophages are specialized immune cells that recognize these abnormal cells and eliminate them by a mechanism called phagocytosis. All normal cells express a protein called CD47 or “don’t eat me signal” to prevent their elimination through phagocytosis. Cancer cells, including leukemic cells, express higher levels of CD47 as a mechanism of protection against macrophage phagocytosis. CD47 blockade leads to an increase in phagocytosis of leukemic cells and better control of the disease. In this review, we explore CD47 function in normal conditions, its role in acute myeloid leukemia progression, and possible ways to block CD47 to enhance elimination of the leukemic cells improving the therapeutic options for patients with acute myeloid leukemia. Abstract CD47 is a surface membrane protein expressed by all normal tissues. It is the so-called “don’t eat me signal” because it protects the cells against phagocytosis. The CD47 interacts with the signal regulatory protein alpha (SIRPα) on the surface of macrophages, leading to downstream inhibitory signaling that dampens phagocytic capacity. Since macrophages exert immune surveillance against cancers, cancer cells overexpress CD47 to defend themselves against phagocytosis. Acute myeloid leukemia (AML) is a cancer of hematopoietic stem/progenitor cells (HSPC), and similar to other types of cancers, leukemic blasts show enhanced levels of CD47. In patients with AML, CD47 has been associated with a higher disease burden and poor overall survival. Blockage of CD47-SIRPα signaling leads to improved phagocytosis of AML cells and better overall survival in xenograft models. However, the introduction of a pro-phagocytic signal is needed to induce greater phagocytic capacity. These pro-phagocytic signals can be either Fc receptor stimulants (such as monoclonal antibodies) or natural pro-phagocytic molecules (such as calreticulin). Based on these pre-clinical findings, various clinical trials investigating the blockade of CD47-SIRPα interaction have been designed as monotherapy and in combination with other anti-leukemic agents. In this review, we will discuss CD47 biology, highlight its implications for AML pathophysiology, and explore the potential clinical translation of disrupting CD47-SIRPα to treat patients with AML.

Leukemia is a complex genetic disease caused by errors in differentiation, growth, and apoptosis of hematopoietic cells in either lymphoid or myeloid lineages. Large-scale genomic characterization of thousands of leukemia patients has produced a tremendous amount of data that have enabled a better understanding of the differences between adult and pediatric patients. For instance, although phenotypically similar, pediatric and adult myeloid leukemia patients differ in their mutational profiles, typically involving either chromosomal translocations or recurrent single-base-pair mutations, respectively. To elucidate the molecular mechanisms underlying the biology of this cancer, continual efforts have been made to develop more contextually and biologically relevant experimental models. Leukemic cell lines, for example, provide an inexpensive and tractable model but often fail to recapitulate critical aspects of tumor biology. Likewise, murine leukemia models of leukemia have been highly informative but also do not entirely reproduce the human disease. More recent advances in the development of patient-derived xenografts (PDXs) or human models of leukemias are poised to provide a more comprehensive, and biologically relevant, approach to directly assess the impact of the in vivo environment on human samples. In this review, the advantages and limitations of the various current models used to functionally define the genetic requirements of leukemogenesis are discussed.

Growing evidence indicates that cell adhesion to extracellular matrix (ECM) plays an important role in cancer chemoresistance. Leukemic T cells express several adhesion receptors of the β1 integrin subfamily with which they interact with ECM. However, the role of β1 integrins in chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL) is still ill defined. In this study, we demonstrate that interactions of human T-ALL cell lines and primary blasts with three-dimensional matrices including Matrigel and collagen type I gel promote their resistance to doxorubicin via β1 integrin. The blockade of β1 integrin with a specific neutralizing antibody sensitized xenografted CEM leukemic cells to doxorubicin, diminished the leukemic burden in the bone marrow and resulted in the extension of animal survival. Mechanistically, Matrigel/β1 integrin interaction enhanced T-ALL chemoresistance by promoting doxorubicin efflux through the activation of the ABCC1 drug transporter. Finally, our findings showed that Matrigel/β1 interaction enhanced doxorubicin efflux and chemoresistance by activating the FAK-related proline-rich tyrosine kinase 2 (PYK2) as both PYK2 inhibitor and siRNA diminished the effect of Matrigel. Collectively, these results support the role of β1 integrin in T-ALL chemoresistance and suggest that the β1 integrin pathway can constitute a therapeutic target to avoid chemoresistance and relapsed-disease in human T-ALL.

Simple Summary Cellular adhesion plays an important role in the development of resistance to chemotherapy (chemoresistance) that represents a major hurdle in the treatment of leukemia and which is a major cause for patient relapse. In this study, we evaluated if cell adhesion to the molecule VCAM-1, which is present in the leukemia microenvironment, can favour the chemoresistance of T acute lymphoblastic leukemia (T-ALL). Our results showed that adhesion of T-ALL cells to VCAM-1 via their receptor VLA-4 induces the resistance of T-ALL cells to doxorubicin by activating the signaling protein PYK2 but not FAK. VLA-4/PYK2 signaling did so by inducing the efflux of doxorubicin. However, adhesion of T-ALL cells to fibronectin via the receptor VLA-5 did not activate PYK2 and had no effect on doxorubicin resistance. These findings suggest that targeting the VLA-4/PYK2 pathway could overcome T-ALL chemoresistance and reduce the risk of patient relapse. Abstract Cell adhesion plays a critical role in the development of chemoresistance, which is a major issue in anti-cancer therapies. In this study, we have examined the role of the VLA-4 integrin, a major adhesion molecule of the immune system, in the chemoresistance of T-ALL cells. We found that attachment of Jurkat and HSB-2 T-ALL cells to VCAM-1, a VLA-4 ligand, inhibits doxorubicin-induced apoptosis. However, their adhesion to fibronectin, which is mainly mediated via VLA-5, had no effect. Even the presence of the chemoattractant SDF1α (Stromal cell-derived factor-1α), which enhances the adhesion of T-ALL cells to fibronectin, did not modify the sensitivity of the cells attached on fibronectin towards doxorubicin-induced apoptosis. Mechanistically, we found that VLA-4 promoted T-ALL chemoresistance by inducing doxorubicin efflux. Our results showed that cell adhesion to both fibronectin and VCAM-1-induced Focal adhesion kinase (FAK) phosphorylation in T-ALL cells. However, only cell adhesion to VCAM-1 led to PYK2 phosphorylation. Inhibition studies indicated that FAK is not involved in doxorubicin efflux and chemoresistance, whereas PYK2 inhibition abrogated both VLA-4-induced doxorubicin efflux and chemoresistance. Together, these results indicate that the VLA-4/PYK2 pathway could participate in T-ALL chemoresistance and its targeting could be beneficial to limit/avoid chemoresistance and patient relapse.

Chromosomal translocations involving the KMT2A gene are among the most common genetic alterations found in pediatric acute myeloid leukemias although the molecular mechanisms that initiate the disease remain incompletely defined. To elucidate these initiating events we used a human model system of acute myeloid leukemia driven by the KMT2A-MLLT3 (KM3) fusion. More specifically, we investigated changes in DNA methylation, histone modifications, and chromatin accessibility at each stage of our model system and correlated these with expression changes. We observed the development of a pronounced hypomethyl - ation phenotype in the early stages of leukemic transformation after KM3 addition along with loss of expression of stem-cell-associated genes and skewed expression of other genes, such as S100A8/9, implicated in leukemogenesis. In addition, early increases in the expression of the lysine demethylase KDM4B was functionally linked to these expression changes as well as other key transcription factors. Remarkably, our ATAC-sequencing data showed that there were relatively few leukemia-specific changes and that the vast majority corresponded to open chromatin regions and transcription factor clusters previously observed in other cell types. Integration of the gene expression and epigenetic changes revealed that the adenylate cyclase gene ADCY9 is an essential gene in KM3-acute myeloid leukemia, and suggested the potential for autocrine signaling through the chemokine receptor CCR1 and CCL23 ligand. Collectively, our results suggest that KM3 induces subtle changes in the epigenome while co-opting the normal transcriptional machinery to drive leukemogenesis.

Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation and clonal expansion of immature myeloblast cells in the blood and bone marrow, leading to hematopoietic failure. Despite the use of aggressive and cytotoxic standard-of-care drugs, patients often relapse and succumb to the disease partially due to the inability of medically unfit patients to withstand the cytotoxic treatments, regrowth from minimal residual disease and the chemo-resistant nature of leukemic stem cells (LSCs) which can remain in a quiescent state and reside in a protective bone marrow niche. Hence, novel therapies targeting unique leukemic stem cell biology are highly needed to eliminate and avoid reoccurrence. High-throughput screens of human AML LSCs are not performed due to technical issues such as low LSC frequency within primary samples, an inability to purify LSCs, and the difficulty maintaining and expanding primary patient samples and LSCs in vitro. We were able to optimize conditions for a 4-week in vitro large-scale expansion (>600 million bulk) of the primary human AML sample 8227 (OCI-AML-8227), functionally validated to be enriched for LSCs in long-term xenotransplant assays (Eppert et al., 2011). These optimized conditions enabled the isolation and maintenance of the LSC-containing fraction for a chemical screen. We isolated the CD34+ LSC-containing fraction (>90% purity) and performed a high-throughput screen of 11,166 chemical molecules using a CellTiter Glo assay followed by a counter screen against normal CD34+ cord blood (CB) hematopoietic stem and progenitor cells. From this HT screen, a total of 61 hits had >70% inhibition on CD34+ 8227 cells and We then performed dose response assays for each candidate compounds and confirmed 35 potent anti-LSC compounds with IC 50 < 1 μM. This refined the types of compounds to including anti-apoptotic inhibitors, GSK inhibitors, protease inhibitors, metabolism inhibitors, HDAC inhibitors, BET inhibitors, nucleic acid synthesis inhibitors, cell cycle inhibitors and Wnt/β-catenin inhibitors. This is interesting as some of the classes of these compounds (inhibitors of GSK, BET, nucleic acid synthesis, Wnt/β-catenin and metabolism) have been shown to target bulk and leukemic stem cells in AML in vitro and in vivo. We now aim to examine LSC eradication in a panel of genetically defined primary AMLs confirmed through in vitro and in vivo assays. Our goal is to be able to understand and establish the molecular mechanisms and biomarkers on primary functional LSCs. Disclosures No relevant conflicts of interest to declare.

Background Benefits of cord blood (CB) transplantation include low rates of relapse and chronic graft-versus-host disease (GVHD). However, CB use has rapidly declined because of the high rates of infections, severe acute GVHD, and transplant-related mortality (TRM) as well as prolonged hospitalization. We therefore initiated a clinical trial with UM171, a potent agonist of hematopoietic stem cell (HSC) self-renewal, to attempt to solve these limitations and permit transplantation of smaller, better HLA-matched CBs. Methods Expansion culture lasted 7 days and patients received a myeloablative conditioning regimen followed by a single CB transplant including both the expanded CD34+ cells and the lymphocyte-containing CD34- fraction. Results Between 9/16-11/18, 22 adults with mostly high- to very high-risk hematologic malignancies were transplanted. Median transplant co-morbidity index was 2 (0-5). Five patients had already failed an allogeneic transplant, 3 patients had acute leukemia not in remission, and 2 patients had relapsed/refractory aggressive lymphoma. UM171 treatment profoundly changed the cellular composition of the graft, including a 600 and 8000-fold increase in dendritic and mast cell progenitors, respectively. Neutrophil engraftment was prompt with median time to 100 and 500 neutrophils/μL of 9.5 and 18 days, respectively. Patients seemed to derive clinical benefit from achieving 100 neutrophils rapidly translating into early resolution of febrile neutropenia (median day 7) contributing to shorter hospitalization when compared to conventional CB transplants at same institution with larger cell doses and similar time to 500 neutrophil engraftment. Median CD4 counts at 3 and 12 months were 218 and 413/μL, respectively. Incidence of grade 3-4 acute GVHD was low (9%) and there was no steroid refractory GVHD or moderate-severe chronic GVHD. More than 90% of patients were off immunosuppressive therapy by 12 months. One patient died of TRM ( Conclusions A 7-day UM171 expansion CB protocol is feasible and provides clinical benefits beyond engraftment, such as very low TRM. If confirmed, UM171 expansion may overcome the shortcomings of CB transplants while maintaining its benefits of low risk of chronic GVHD and relapse. We have now embarked on a phase 2 trial to confirm if a UM171 modified graft, enriched in dendritic cells, will have a potent antileukemia effect in extremely high-risk patients such as refractory leukemias and high risk mutations (e.g.: p53, EVI-1).

Chemoresistance is a major hurdle in anti-cancer therapy. Growing evidence indicates that integrin-mediated cell adhesion to extracellular matrix plays a major role in chemoresistance. However, the underlying mechanisms are not fully understood. We have previously shown that the collagen-binding integrin α2β1 promoted doxorubicin resistance in acute T cell lymphoblastic leukemia (T-ALL). In this study, we found that acute myeloid leukemia (AML) cell lines also express α2β1 integrin and collagen promoted their chemoresistance as well. Furthermore, we found that high levels of α2 integrin correlate with worse overall survival in AML. Our results showed that doxorubicin-induced apoptosis in leukemic cells is associated with activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and that collagen inhibited this pathway. The protective effect of collagen is associated with the inhibition of Rac1-induced DNA damage as evaluated by the comet assay and the phosphorylated levels of histone H2AX (γ-H2AX). Together these results show that by inhibiting pro-apoptotic Rac1, α2β1 integrin can be a major pathway protecting leukemic cells from genotoxic agents and may thus represent an important therapeutic target in anti-cancer treatment.

FLT3, DNMT3A, and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56highCD34low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (HLF). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34+GPR56+ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 (HES1) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup.

Acute myeloid leukemias (AMLs) result from a series of genetic events occurring in a stem or progenitor hematopoietic cell that gives rise to their clonal expansion and an impaired capacity to differentiate. To circumvent the genetic heterogeneity of AML patient cohorts, we have developed a model system, driven by the MLL-AF9 (MA9) oncogene, to generate multiple human leukemias using progenitor cells from a single healthy donor. Through stepwise RNA-sequencing data generated using this model and AML patients, we have identified consistent changes associated with MA9-driven leukemogenesis and demonstrate that no recurrent secondary mutations are required. We identify 39 biomarkers whose high expression level is specific to this genetic subtype of AML and validate that many of these have diagnostic utility. We further examined one biomarker, the receptor tyrosine kinase (RTK) RET, and show through shRNA knockdowns that its expression is essential for in vivo and in vitro growth of MA9-AML. These results highlight the value of novel human models of AML derived from single donors using specific oncogenic fusions to understand their biology and to uncover potential therapeutic targets.

Acute myeloid leukemia (AML) is a disease that results from the uncontrolled growth of primitive myeloid progenitor cells that are unable to undergo terminal differentiation. To better understand the genetic and epigenetic changes involved in leukemogenesis, we use a human model leukemia system where cord blood donor cells from a single donor are transduced with the human KMT2A-MLLT3 gene fusion to produce leukemias. We have generated multiple model leukemias and compared these to genetically matched pediatric AML patient samples that has allowed us to characterize the step-wise epigenetic and splicing changes that accompany leukemogenesis, allowing us to build an integrated view of this process. Our analysis of these data have revealed a number of candidate biomarker genes that are expressed in KMT2A translocated AMLs but not normal blood cells, some of which are important for leukemic growth. These candidate genes, which have validated diagnostic value, have also been used as targets for monoclonal antibody generation in order to assess their value as potential immunotherapeutics. Having established the value of this approach, we are now expanding our models to include additional oncogenic drivers in order to elucidate the differences in patient outcomes associated with the presence of specific fusions. Moreover, we have recently leveraged these model leukemias and matched patient samples to perform a high-throughput small molecule screen to identify novel therapeutic vulnerabilities and deconvolute the role of secondary mutations in pediatric patients. The establishment of this chemogenomic pipeline to generate, characterize, and then screen model leukemias will provide not only insight into the molecular mechanisms involved in this disease, but also how they can be rationally targeted to improve patient outcomes.

Next generation DNA sequencing has provided significant insights into the genetic determinants of acute myeloid leukemia (AML). Large scale sequencing studies of AML patient cohorts have revealed a remarkable level of genetic heterogeneity between patients who nevertheless have the same disease phenotype. As a solution to the problem of extensive genetic diversity between patients, we have modified a previously published model system in order to generate multiple human leukemias from CD34+ cord blood cells from a single healthy donor. A human MLL-AF9 (MA9) fusion gene is retrovirally introduced into the donor cells which are then cultured for 30 days before being transplanted into immunocompromised (NSG) mice that subsequently develop either AML or acute B-cell lymphoblastic leukemia (B-ALL) after ~24 weeks. We have now generated 22 leukemias from 4 single donors and have performed RNA-seq on the samples during their step-wise leukemic transformation (i.e. CD34+ cells, CD34+ cells with MA9, and the resulting leukemias). We have compared these data to RNA-seq data we have generated for several pediatric AML patients with MA9 translocations, as well as normal blood cells and other tissues. This analysis has revealed 39 candidate genes with an expression pattern highly specific for MA9 AMLs. Interestingly, we can find no evidence for secondary mutations acquired by the human leukemias in our model system, suggesting the human MA9 translocation is sufficient to generate leukemias in this context. To understand the epigenetic impact of the MA9 fusion gene, we have examined the DNA methylation changes at each stage in our model system using a capture survey approach (e.g. Human Methyl-Seq; Agilent) and used this same approach for several primary patient samples with MLL translocations. These data have been correlated with gene expression changes within the model system and have revealed a number of specific changes with relevance for the process of transformation. Lastly, functional assessment of specific candidate genes through shRNA knock-down experiments has shown that at least some of these candidate genes, which are known oncogenes in other tumor types, are essential for MA9 AML. In summary, the combination of RNA-seq data from patient and single donor model AMLs has highlighted consistent genetic changes associated with this AML sub-group, and has also revealed novel potential therapeutic targets. Citation Format: Frederic Barabe, Magalie Celton, Audrey Forest, Anne Bergeron, Radia Johnson, Laurine Gil, Angélique Bellemare-Pelletier, Sonia Cellot, Josee Hebert, Etienne Gagnon, Brian T. Wilhelm. Integrated genetic and epigenetic analysis of model and patient acute myeloid leukemias. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-14.

Using next-generation sequencing of primary acute myeloid leukemia (AML) specimens, we identified to our knowledge the first unifying genetic network common to the two subgroups of KMT2A (MLL)-rearranged leukemia, namely having MLL fusions or partial tandem duplications. Within this network, we experimentally confirmed upregulation of the gene with the most subtype-specific increase in expression, LOC100289656, and identified cryptic MLL fusions, including a new MLL-ENAH fusion. We also identified a subset of MLL fusion specimens carrying mutations in SPI1 accompanied by inactivation of its transcriptional network, as well as frequent RAS pathway mutations, which sensitized the leukemias to synthetic lethal interactions between MEK and receptor tyrosine kinase inhibitors. This transcriptomics-based characterization and chemical interrogation of human MLL-rearranged AML was a valuable approach for identifying complementary features that define this disease.

Acute myeloid leukemias (AMLs) with translocations of the mixed lineage leukemia (MLL/KMT2A) gene are common in young patients and are generally associated with poor clinical outcomes. The molecular biology of MLL fusion genes remains incompletely characterized and is complicated by the fact that more than 100 different partner genes have been identified in fusions with MLL. The continuously growing list of MLL fusions also represents a clinical challenge with respect to identification of novel fusions and tracking of the fusions to monitor progression of the disease after treatment. Recently, we have developed a novel single-donor model leukemia system that permits the development of human AML from normal cord blood cells. Gene expression analysis of this model and of MLL-AML patient samples has identified a number of candidate biomarker genes with highly biased expression on leukemic cells. Here, we present data demonstrating the potential clinical utility of several of these candidate genes for identifying known and novel MLL fusions.

Multipotent long-term repopulating hematopoietic stem cells (LT-HSCs) can self-renew or differentiate into the less primitive short-term repopulating stem cells (ST-HSCs), which themselves produce progenitors that ensure the daily supply of all essential blood components. The Polycomb group (PcG) protein BMI1 is essential for the activity of both HSCs and progenitor cells. Although BMI1 operates by suppressing the Ink4a/Arf locus in progenitors and ST-HSCs, the mechanisms through which this gene regulates the activity of LT-HSCs remain poorly understood. Toward this goal, we isolated BMI1-containing protein complexes and identified UBAP2L as a novel BMI1-interacting protein. We also showed that UBAP2L is preferentially expressed in mouse and human HSC-enriched populations when compared with more mature cell types, and that this gene is essential for the activity of LT-HSCs. In contrast to what is observed for Bmi1 knockdown, we found that UBAP2L depletion does not affect the Ink4a/Arf locus. Given that we demonstrated that BMI1 overexpression is able to rescue the deleterious effects of Ubap2l downregulation on LT-HSC activity and that UBAP2L is part of a PcG subcomplex comprising BMI1, we propose a model in which at least 2 different BMI1-containing PcG complexes regulate HSC activity, which are distinguishable by the presence of UBAP2L.

Background Benefits of cord blood (CB) transplantation include low rates of relapse and chronic graft-versus-host disease (cGVHD). However, CB use is rapidly declining because of delayed neutrophil recovery, high rates of infections and severe acute GVHD leading to high transplant-related mortality (TRM) and prolonged hospitalization. Several of these complications are related to the low cell number of CB grafts, compromising early engraftment and optimal HLA matching. In order to improve these limitations, we initiated a phase II clinical trial exploiting UM171 expanded CB. This molecule has previously been shown to efficiently expand stem and progenitor cells in 7-day ex vivo cultures. More recent studies have also documented the dominant expanding effect of UM171 on several immuno-modulatory cells, thus profoundly changing graft composition and possibly further reducing relapse and GVHD. Methods Patients (pts) received a myeloablative conditioning regimen. On day (D)-7 of transplant, CB was thawed and CD34+ selected. The CD34- lymphocyte containing fraction was cryopreserved and infused on D+1. The CD34+ component was placed in a closed culture system with UM171 and media was injected once a day until D0, when cells were washed and infused. This fed-batch culture system allowed for small culture volumes, saving cost and labor. Moreover, the short duration of expansion made the process practical and clinically viable. Findings Between 9/16-11/18, 22 adults with a median age of 44 years and high- to very high-risk hematologic malignancies were transplanted with a single UM171 CB. Five pts (23%) had already failed a prior allogeneic transplant and 5 (23%) had refractory/relapsed acute leukemia or aggressive lymphoma. Median co-morbidity index was 2 and 36% of pts had an index ≥3. Because minimal cell dose requirements were lower, we had access to 47% of the CBs in the banks instead of 5% for a 70 kg pt. Consequently, >80% of pts received a ≥6/8 HLA matched CB. UM171 profoundly changed graft composition, including a 600 and 8000-fold increase in dendritic and mast cells, respectively. Median 1st day of 100 and 500 neutrophils were D+10 and D+18, respectively. Achieving 100 neutrophils was 5 days faster than seen with pts receiving peripheral blood (PB) or marrow (BM) at same institution and appeared cell dose independent, suggesting that clinically meaningful expansion of an early repopulating myeloid progenitor is at saturation even with smaller CBs. In contrast, attaining 500 neutrophils was accelerated but dependent on cell dose and similar to our BM-PB pts. More importantly, pts appeared to derive clinical benefit beyond neutrophil engraftment as median last day of fever was D+8, much earlier than engraftment and 4 days earlier than seen with our PB-BM pts, translating into a shorter duration of admission, similar to that with BM-PB. Median CD4 count was 218/μL and 413/μL at 3 and 12 months, respectively. Cumulative incidences (CI) of grade 2-4 and 3-4 acute GVHD were 60% and 9%, respectively. We observed no steroid refractory acute GVHD, no moderate/severe cGVHD and 91% of patients were off immunosuppressive therapy by 12 months. One patient died of TRM ( Interpretation A 7-day UM171 expansion CB protocol is feasible and provides clinical benefits beyond engraftment, such as very low TRM and severe GVHD with prompt withdrawal of immunosuppression resulting in excellent GRFS. Hypotheses to explain this could be earlier achievement of 100 neutrophils, protective effect of the immuno-modulatory dendritic cells on mucosal integrity and relapse, and mast cells on gut GVHD, as well as better HLA matching. If confirmed, UM171 expansion may overcome the shortcomings of CB transplants while maintaining its benefits of low risk of cGVHD and relapse. Encouraged by our observation that several pts with very high-risk disease (e.g. refractory leukemia) remain in CR, we have now embarked on a Canadian and US trial to ascertain if indeed UM171 modified grafts will have a potent antileukemia effect in extremely high-risk disease pts such as those with refractory acute leukemias and high-risk molecular anomalies like p53 mutation and EVI-1 rearrangement. Figure Disclosures Cohen: ExCellThera: Consultancy, Equity Ownership, Patents & Royalties: Royalities from sales of UM171, Research Funding. Roy:Celgene: Consultancy, Honoraria, Research Funding; ExCellThera: Patents & Royalties: Royalties from sales of UM171, Research Funding; Amgen Canada: Honoraria; Janssen Canada: Honoraria; Sanofi Canada: Research Funding. Delisle:ExCellThera: Patents & Royalties, Research Funding. Bambace:ExCellThera: Patents & Royalties: royalities. Ahmad:ExCellThera: Patents & Royalties. Lachance:ExCellThera: Patents & Royalties. Bernard:ExCellThera: Patents & Royalties. Kiss:ExCellThera: Patents & Royalties. Busque:Paladin: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; ExCellThera: Patents & Royalties; BMS: Consultancy. Roy:Kiadis Pharma: Other: Travel support; University of Montreal: Patents & Royalties: Author on patent; Hopital Maisonneuve-Rosemont: Patents & Royalties: Author on patent. Milano:ExCellThera: Research Funding; Amgen: Research Funding. Sauvageau:ExCellThera: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. OffLabel Disclosure: None of the medications listed are approved for this indication.

S100A8 and S100A9 are calcium-binding proteins predominantly expressed by neutrophils and monocytes and play key roles in both normal and pathological inflammation. Recently, both proteins were found to promote tumor progression through the establishment of premetastatic niches and inhibit antitumor immune responses. Although S100A8 and S100A9 have been studied in solid cancers, their functions in hematological malignancies remain poorly understood. However, S100A8 and S100A9 are highly expressed in acute myeloid leukemia (AML), and S100A8 expression has been linked to poor prognosis in AML. We identified a small subpopulation of cells expressing S100A8 and S100A9 in AML mouse models and primary human AML samples. In vitro and in vivo analyses revealed that S100A9 induces AML cell differentiation, whereas S100A8 prevents differentiation induced by S100A9 activity and maintains AML immature phenotype. Treatment with recombinant S100A9 proteins increased AML cell maturation, induced growth arrest, and prolonged survival in an AML mouse model. Interestingly, anti-S100A8 antibody treatment had effects similar to those of S100A9 therapy in vivo, suggesting that high ratios of S100A9 over S100A8 are required to induce differentiation. Our in vitro studies on the mechanisms/pathways involved in leukemic cell differentiation revealed that binding of S100A9 to Toll-like receptor 4 (TLR4) promotes activation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2, and Jun N-terminal kinase signaling pathways, leading to myelomonocytic and monocytic AML cell differentiation. These findings indicate that S100A8 and S100A9 are regulators of myeloid differentiation in leukemia and have therapeutic potential in myelomonocytic and monocytic AMLs.

Amplification of the human epidermal growth factor receptor 2 (HER2) gene is associated with worse prognosis and decreased overall survival in breast cancer patients. The HER2 gene contains several polymorphisms; two of the best-characterized HER2 polymorphisms are Ile655Val and Ala1170Pro. The aim of this study was to evaluate the association between these two HER2 polymorphisms in normal breast and breast cancer tissues and known breast cancer prognostic factors in a retrospective cohort study of 73 women with non-metastatic HER2-positive breast cancer. HER2 polymorphisms were assessed in breast cancer tissue and normal breast tissue using TaqMan assay. Ala1170Pro polymorphism in normal breast tissue was associated with age at diagnosis (p = 0.007), tumor size (p = 0.004) and lymphovascular invasion (p = 0.06). Similar significant associations in cancer tissues were observed. No association between the Ile655Val polymorphism and prognostic factors were observed. However, we found significant differences in the distribution of Ile655Val (p = 0.03) and Ala1170Pro (p = 0.01) genotypes between normal breast and breast tumor tissues. This study demonstrates that only the Ala1170Pro polymorphism is associated with prognostic factors in HER2-positive breast cancer patients. Moreover, our results suggest that both HER2 polymorphisms could play a significant role in carcinogenesis in non-metastatic HER2-positive breast cancer women.

Interleukin-15 deficiency promotes the development of T-cell acute lymphoblastic leukemia in non-obese diabetes mice with severe combined immunodeficiency

Background: Trastuzumab is well tolerated without major side effects except for cardiac toxicity. Although a number of clinical parameters have been associated with trastuzumab-associated cardiac toxicity (TACT), there is some indication that genetic variation of the HER2 gene may be associated with TACT in a population of metastatic breast cancer patients. However, this finding needs confirmation and we looked at a population of non-metastatic breast cancer. Purpose: This study aimed to evaluate the association between cardiac toxicity and HER2 [Ile655Val] polymorphism in non-metastatic breast cancer patients treated with trastuzumab. Method: The Ile655Val HER2 polymorphism was assessed in 41 women using TaqMan technology. For this study, the genotyping was performed using DNA extracted from normal breast tissue located at more than 1 cm of any other lesions. Charts review was used to collect information on TACT which was defined as a decrease in the left ventricular ejection fraction (LVEF) of ≥15% or ≥10% with a resulting LVEF < 50% or any follow-up LVEF of < 45%. The Fisher exact test was used to evaluate the association between cardiac toxicity and HER2 polymorphism. Results: No deviation from the Hardy-Weinberg equilibrium has been observed for the allele and genotype frequencies. The distribution of HER2 polymorphism was 10 Ile/Val (24%) and 31 Ile/Ile (76%). In this population, 22% (9/41) developed a cardiac toxicity. The Val655Ile genotype was associated with cardiac toxicity (0.03). In this preliminary study, 50% (5/10) of Ile/Val carriers compared to 13% (4/31) of Ile/Ile carriers showed TACT. Conclusion: HER2 Ile655Val polymorphism may be an efficient marker of TACT considering this tendency with this small cohort of patients. Larger sample is needed to strengthen this conclusion, since this result may influence on prescribing decision for adjuvant chemotherapy and anti-HER2 therapy in HER2 positive patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-12.

Cord blood (CB) transplants have fallen into disfavor in large part due to low cell dose leading to prolonged hospitalizations and high transplant related mortality (TRM). UM171, a novel and potent agonist of hematopoietic stem cell (HSC) self-renewal could solve this major limitation, allowing for CB's important qualities of lower risk of chronic GVHD and relapse to prevail. In addition, UM171 could permit transplantation of smaller, better HLA matched cords, associated with lower TRM. Hence, we initiated a clinical trial to test the safety and efficacy of UM171 expanded CB (eCB). Our goal was to design a clinically viable eCB transplant with a TRM as low or lower than other HSC sources all the while maintaining CB's low relapse rate. Patients (pts) received a myeloablative conditioning regimen. On day (D)-7 of transplant, CB was thawed and CD34+ selected. The CD34- lymphocyte containing fraction was cryopreserved and infused on D+1. The CD34+ component was placed in a closed culture system with UM171 and media was injected once a day until D0, when cells were washed and infused. This fed-batch culture system allowed for small culture volumes, saving cost and labor. Between 7/16-6/18, 21 adult pts (median age 44 years) were transplanted with an eCB. Median final culture volume and net viable CD34 fold expansion were 670 mL and 35, respectively. Median 1st day of 100 and 500 neutrophils were D+10 and D+18, respectively. Achieving 100 neutrophils was 5 days faster than seen with our pts receiving peripheral blood (PB) or marrow (BM) and appeared cell dose independent, suggesting that clinically meaningful expansion of an early repopulating myeloid progenitor is at saturation even with smaller CBs. In contrast, attaining 500 neutrophils was accelerated but dependent on cell dose. More importantly, pts appeared to derive clinical benefit beyond neutrophil engraftment (defined as 500 neutrophils). Pts' median last day of fever prior to 500 neutrophils was D+8, much earlier than engraftment and 4 days earlier than seen with our PB-BM pts. We offer 2 hypotheses as explanation: i) 100 neutrophils, which are attained early, provide significant defense against infection, ii) the graft contains a significant proportion of dendritic cell precursors (30-40%) which offer mucosal protection during severe neutropenia. Duration of hospitalization was shorter by 12 days and longer by 2 days compared to our non eCB and PB-BM transplants, respectively. In addition, because cell dose requirements were lower, 12/21 pts received a better HLA matched CB, thus >80% of patients were transplanted with a ≥6/8 HLA matched eCB. As a result of lower minimal cell dose criteria, we can now use ∼half the CBs in the banks instead of only 5% for a 70 kg patient. Platelet engraftment occurred at a median of 42 days. With a median follow up of 14 months, there has been no CMV disease, no PTLD, 2 adenovirus cystitis, 2 (10%) grade 3-4 acute GVHD, no moderate/severe chronic GVHD and 1 TRM (5%) despite a median comorbidity index of 2 (0-5). Full donor chimerism was achieved in all cell subsets. Immune recovery was faster than seen in our unrelated donor transplants who routinely receive ATG prophylaxis with 196, 300 and 413 CD4+/µL at 3, 6 and 12 months, respectively. Interestingly, transcriptome analysis of UM171-eCB cells shows an enhanced lymphoid progenitor-associated gene signature when compared to DMSO exposed cells. Animals transplanted with UM171-eCB cells showed a 20 to 35-fold increase in thymic cellularity at 8 weeks post-transplant. Despite some very high risk pts in our trial, only 3 relapsed. Overall, progression free, and GVHD/relapse free survival (GRFS) are excellent at 95, 77 and 67%, respectively, at 12 months. A 7 day UM171 single eCB protocol is feasible and provides clinical benefits beyond faster engraftment with fewer infectious complications, better HLA matching and very low TRM, all the while saving production and hospitalization costs. Nevertheless, longer follow up will be required to better assess relapse howbeit encouraging preliminary results. Furthermore, patients' quality of life is paramount and best evaluated by GRFS which is excellent thanks to a very low rate of significant chronic GVHD all the while maintaining a low risk of relapse. In conclusion, this 1st trial documents the potency of UM171 and positions UM171-eCB as a promising HSC source which could compete with the current standard of care. Figure. Figure. Disclosures Cohen: ExCellThera: Patents & Royalties: Royalities from sales of UM171. Roy:ExCellThera: Patents & Royalties: Royalities from sales of UM171. Lachance:ExCellThera: Patents & Royalties: Royalities from sales of UM171. Roy:Hopital Maisonneuve Rosemont: Patents & Royalties: Author on patent; Kiadis Pharma: Other: Travel support; University of Montreal: Patents & Royalties: Author on patent. Busque:BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Paladin: Consultancy. Kiss:Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka: Membership on an entity's Board of Directors or advisory committees, Research Funding. Caudrelier:ExCellThera: Employment. Zandstra:ExCellThera: Equity Ownership. Sauvageau:ExCellThera: Employment, Equity Ownership.

The hematopoietic system produces appropriate levels of blood cells over an individual's lifetime through a careful balance of differentiation, proliferation and self-renewal. The acquisition of genetic and epigenetic alterations leads to deregulation of these processes and the development of acute leukemias. A prerequisite to targeted therapies directed against these malignancies is a thorough understanding of the processes that subvert the normal developmental program of the hematopoietic system. This involves identifying the molecular lesions responsible for malignant transformation, their mechanisms of action and the cell type(s) in which they occur. Over the last 3 decades, significant progress has been made through the identification of recurrent genetic alterations and translocations in leukemic blast populations, and their subsequent functional characterization in cell lines and/or mouse models. Recently, primary human hematopoietic cells have emerged as a complementary means to characterize leukemic oncogenes. This approach enables the process of leukemogenesis to be precisely modeled in the appropriate cellular context: from primary human hematopoietic cells to leukemic stem cells capable of initiating disease in vivo. Here we review the model systems used to study leukemogenesis, and focus particularly on recent advances provided by in vitro and in vivo studies with primary human hematopoietic cells.

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy, which is initiated and driven by a rare fraction of leukemia stem cells (LSCs). Despite the difficulties of identifying a common LSC phenotype, there is increasing evidence that high expression of stem cell gene signatures is associated with poor clinical outcome. Identification of functionally distinct subpopulations in this disease is therefore crucial to dissecting the molecular machinery underlying LSC self-renewal. Here, we combined next-generation sequencing technology with in vivo assessment of LSC frequencies and identified the adhesion G protein-coupled receptor 56 (GPR56) as a novel and stable marker for human LSCs for the majority of AML samples. High GPR56 expression was significantly associated with high-risk genetic subgroups and poor outcome. Analysis of GPR56 in combination with CD34 expression revealed engraftment potential of GPR56(+)cells in both the CD34(-)and CD34(+)fractions, thus defining a novel LSC compartment independent of the CD34(+)CD38(-)LSC phenotype.

The signal transduction mechanisms associated with the ligation of FcγRIIA in human neutrophils are as yet only incompletely characterized. In the present study, we have investigated the distribution and fate of FcγRIIA following its cross-linking. The results obtained indicate that cross-linking of FcγRIIA led, within a few seconds, to its translocation into a nonionic detergent-insoluble fraction. This was followed, within a couple of minutes, by a substantial loss of immunoreactive FcγRIIA in the cells. The stimulated degradation of FcγRIIA was blocked by the Src kinase inhibitor PP1 but not by wortmannin, ST-638, piceatannol, or cytochalasin B. Cross-linked FcγRIIA could be solubilized by saponin (in the presence of Nonidet P-40) and by β-octylglucoside. Sucrose gradient analysis of the distribution of FcγRIIA revealed that its cross-linking led to its translocation into the pellets and not the light buoyant density fractions classically associated with lipid rafts. Disruption of cholesterol-containing membrane microdomains with filipin prevented the degradation of FcγRIIA but did not inhibit the stimulation of the pattern of tyrosine phosphorylation or the mobilization of calcium that followed FcγRIIA cross-linking. These data suggest that both cholesterol-rich domains and Src kinases are required for the degradation of the activated FcγRIIA and provide new insights into the early events following FcγRIIA cross-linking.

The effects of cholesterol-perturbing agents on the mobilization of calcium induced upon the stimulation of human neutrophils by chemotactic factors were tested. Methyl-beta-cyclodextrin and filipin did not alter the initial peak of calcium mobilization but shortened the duration of the calcium spike that followed the addition of fMet-Leu-Phe. These agents also inhibited the influx of Mn(2+) induced by fMet-Leu-Phe or thapsigargin. Methyl-beta-cyclodextrin and filipin completely abrogated the mobilization of calcium induced by 10(-10) m platelet-activating factor, which at this concentration depends to a major extent on an influx of calcium as well as the influx of calcium induced by 10(-7) m platelet-activating factor. On the other hand, methyl-beta-cyclodextrin and filipin enhanced the mobilization of calcium induced by ligation of FcgammaRIIA, an agonist that did not induce a detectable influx of calcium. Finally, methyl-beta-cyclodextrin and filipin enhanced the stimulation of the profile of tyrosine phosphorylation, the activity of phospholipase D (PLD), and the production of superoxide anions induced by fMet-Leu-Phe. These results suggest that the calcium channels utilized by chemotactic factors in human neutrophils are either located in cholesterol-rich regions of the plasma membrane, or that the mechanisms that lead to their opening depend on the integrity of these microdomains.

The effects of soluble and particulate agonists on the tyrosine phosphorylation levels of the proto-oncogene Cbl in human neutrophils were examined. Experimental conditions allowing the maintenance of Cbl as well as of its tyrosine phosphorylation status were first established. Their use allowed us to observe that Cbl was tyrosine phosphorylated in response to some (FcγRII ligation, opsonized bacteria and zymosan, granulocyte-macrophage colony-stimulating factor, monosodium urate, and calcium pyrophosphate microcrystals), but not all (fMet-Leu-Phe, interleukin-8) neutrophil agonists. Cbl was also shown to account for a varying proportion of the 120-kDa phosphoprotein(s) observed in response to the above stimuli. These data establish that Cbl is present in human neutrophils and that its level of tyrosine phosphorylation is modulated by some of these cells' agonists, and in particular by phagocytic particles. Furthermore, the signaling pathways activated by chemotactic factors and the other neutrophil stimuli tested in this investigation diverge at or downstream from the tyrosine phosphorylation of Cbl.

Activation of various cell types by different agonists is known to stimulate a transient increase in the level of tyrosine phosphorylation of certain cellular proteins. Such phosphorylation is essential for mediating signalling by these agonists. The preservation of the tyrosine phosphorylation of proteins in lysates has proven to be a difficult task in neutrophils because of their large arsenal of proteases and phosphatases. Here we describe a technique that we found useful for preserving the tyrosine phosphorylation of cellular proteins. The technique depends on the denaturing lysis of neutrophils followed by the removal of the denaturing agents using Sephadex columns. Preparing neutrophil lysates by this technique has proven to be reliable in terms of maintaining the stability of the tyrosine phosphorylated proteins of various molecular weights and their subsequent immunoprecipitation and identification.

Trastuzumab has no major side-effects except the potential for cardiac toxicity. The main objective of this study was to evaluate the association between trastuzumab-associated cardiac toxicity and two potential risk factors: alcohol intake and HER2 polymorphisms.In a retrospective cohort study of 237 women with non-metastatic HER2-positive breast cancer treated with trastuzumab, traditional risk factors were assessed by review of medical records, alcohol use by an administered questionnaire to women (n=132), and HER2 polymorphisms (Ile655Val and Ala1170Pro) using TaqMan assays (n=73).Association was observed between alcohol intake (10 drinks and more per week) during the trastuzumab treatment and cardiac toxicity (p=0.04). For polymorphisms, compared to Ile/Ile carriers, HER2 Ile/Val was associated with a higher risk of cardiac toxicity (p=0.02).Heavy alcohol use during the course of trastuzumab treatment and the HER2 Ile/Val genotype may constitute risk factors for cardiac toxicity.

Dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor expressed at high levels on dendritic cells (DCs). This surface molecule acts as an attachment factor for HIV-1 on DCs and contributes to trans- and cis-infection pathways. Moreover, DICR is induced by HIV-1 in CD4+ T cells and promotes virus replication in this cell type. Nothing is known hitherto about the DCIR-dependent signaling, which is induced following HIV-1 ligation. First, specific pharmacologic inhibitors were tested on HIV-1 binding/entry and, second, specific antisense oligonucleotides targeted, more specifically kinases and phosphatases, were used. Our results show that SHP-1, SHP-2, Syk, and Src kinases (ie, Src, Fyn, and Hck) as well as PKC-α and MAP kinases (ie, Erk1/2 and p38) are all involved in the DCIR-mediated signal transduction pathway triggered by HIV-1. By mutagenesis and through the use of intracellular phosphorylated peptides, we show as well a pivotal role for the tyrosine and threonine residues of the DCIR immunoreceptor tyrosine-based inhibitory motif (ITIM). Our data suggest for the first time an involvement of ITIM domain in HIV-1–mediated signaling events and a relationship between phosphorylation events and DCIR function with respect to HIV-1 biology.

Kelly et al. (Brevia, 20 July 2007, p. 337) questioned xenotransplant experiments supporting the cancer stem cell (CSC) hypothesis because they found a high frequency of leukemia-initiating cells (L-IC) in some transgenic mouse models. However, the CSC hypothesis depends on prospective purification of cells with tumor-initiating capacity, irrespective of frequency. Moreover, we found similar L-IC frequencies in genetically comparable leukemias using syngeneic or xenogeneic models.

Our understanding of leukemia development and progression has been hampered by the lack of in vivo models in which disease is initiated from primary human hematopoietic cells. We showed that upon transplantation into immunodeficient mice, primitive human hematopoietic cells expressing a mixed-lineage leukemia ( MLL ) fusion gene generated myeloid or lymphoid acute leukemias, with features that recapitulated human diseases. Analysis of serially transplanted mice revealed that the disease is sustained by leukemia-initiating cells (L-ICs) that have evolved over time from a primitive cell type with a germline immunoglobulin heavy chain (IgH) gene configuration to a cell type containing rearranged IgH genes. The L-ICs retained both myeloid and lymphoid lineage potential and remained responsive to microenvironmental cues. The properties of these cells provide a biological basis for several clinical hallmarks of MLL leukemias.

Insights into the complex clonal architecture of acute myeloid leukemia (AML) unravelled by deep sequencing technologies have challenged the concept of AML as a hierarchically organised disease initiated and driven by rare self-renewing leukemic stem cells (LSCs). In contrast to normal human hematopoietic stem cells (HSCs), which are highly enriched in the CD34+ CD38- population, LSCs have also been found in the CD34- and the CD38+ fractions questioning the existence of a consistent LSC surface marker profile for AML. Besides, low LSC frequencies in primary samples, rapid onset of differentiation upon ex vivo culture, and genetic inter-specimen heterogeneity hamper the dissection of the molecular machinery that drives LSC self-renewal. We performed RNA-Sequencing of primary human AML samples and assessed LSC frequencies by limiting dilution analyses for 56 of these in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. By comparing gene expression profiles between high vs low LSC frequency leukemias, we identified the G-protein coupled receptor 56 (GPR56) has significantly more expressed in high LSC frequency leukemias. We validated the RNA-seq data with protein expression by FACS and found an excellent correlation. To determine whether GPR56 positive cells overlapped with the known LSC-associated phenotype CD34+ CD38-, we stained 45 AML samples with CD34, CD38, GPR56, and antibodies against other described LSC markers. Although CD34+ GPR56+ and CD34+ CD38- compartments identified the same population in some samples, we found in the majority of samples that GPR56 further subdivided the CD34+ CD38- compartment. Accordingly, not only the proportions of total GPR56+ and CD34+ GPR56+ cells were significantly higher in LSChigh versus LSClow samples, but also the proportion of GPR56+ cells within the CD34+ CD38- compartment was significantly different between the groups indicating that GPR56 might be of additional value to what is currently considered the best described LSC phenotype. The percentage of total CD34 positive cells did not correlate with LSC frequency clearly distinguishing GPR56 from CD34 or CD38, which are only suitable LSC markers when used in combination. We analysed other potential LSC markers (TIM3, CD96, CD44, CD123, CLL1 and CD47) in our RNA-Seq dataset and by FACS analysis in combination with CD34 as we did for GPR56 and none of them correlated with LSC frequency in our sample collection. To determine whether GPR56 discriminates engrafting LSCs from non-LSCs, we sorted GPR56+ and GPR56- cells within the CD34-positive and -negative compartments from selected specimens with known engraftment potential. We found that GPR56 identified the engrafting fraction in CD34positive AML samples, with a >50 fold enrichment in LSC in the CD34+GRP56+ fraction vs the CD34+GPR56- fraction within the same sample, demonstrating that GPR56 is a good LSC marker. Specimens with high molecular or cytogenetic risk such as chromosome 5 or 7 anomalies and EVI1- rearrangementexpressed high levels of both, GPR56 and CD34, while samples with coexistent FLT3 -ITD, DNMT3A, and NPM1 mutations displayed a unique CD34low GPR56high profile. Moreover, we found a divergent distribution of variant allele frequencies in GPR56+ versus GPR56- fractions identifying GPR56 as a discriminator of leukemic sub-clones with high and low NSG engrafting capacity. Analysis of engrafted cells re-sorted based on GPR56 after being harvested from mouse bone marrow revealed reduced complexity of the clonal composition. Most importantly, GPR56 positive cells differentiated to GPR56 negative cells in mice, which did not happen in the human niche, in which GPR56 positive and negative fractions represented two independently evolved subclones. In summary our work identifies GPR56 as a novel LSC marker in AML and also shows that GPR56 readily identifies a functionally distinct LSC-rich subclone in the majority of human AML patients and reveals hitherto unforeseen complexity in the interaction between human LSCs and the NSG mouse environment. Disclosures No relevant conflicts of interest to declare.

The myeloid-related proteins S100A8 (MRP8) and S100A9 (MRP14) are endogenous alarmins abundantly and constitutively expressed by myeloid cells (neutrophils, monocytes and immature myeloid cells). S100A8 and S100A9 proteins exist as homodimers but also associate to form the heterodimer calprotectin (S100A8/A9) and are up-regulated in several inflammatory diseases and human cancers. In patients with acute myeloid leukemia (AML), the concentration of S100A8/A9 in serum is elevated and the expression of S100A8 correlates with poor prognosis. However, the role of these proteins in hematologic malignancies is largely unknown. Here, we studied the roles of S100A8 and S100A9 in a mouse model ofAML induced by overexpression of Hoxa9 and Meis1 (H9M1). As observed in human, mice developing AML have a substantial increase in S100A8/A9 their serum (5.0µg/ml ± 1.0µg/mL vs 0.5µg/mL ± 0.1µg/mL for the control, p To investigate if secreted S100A8 and S100A9 proteins play a role in leukemogenesis, H9M1 AML were transplanted to secondary recipients were treated intraperitoneally (i.p) with anti-S100A8 or anti-S100A9 antibodies. Blocking S100A8 led to a marked delay in leukemia progression and significantly extended survival compared to control immunoglobulins (IgG) (31 days vs 39.5 days, p=0.010) with an increase of the CD11b+ Gr-1+ double positive population (78.8%±1.4 vs 90.1%±3, p=0.038). In contrast, no differences in overall survival were observed between control IgG and anti-S100A9 treated mice. In addition, we demonstrate that anti-S100A8 treatments reduced AML cell proliferation through the G0/G1 cell cycle arrest. Thus, blocking S100A8 reduces leukemogenesis and induced leukemic blast maturation in AML. To further investigate the roles of S100A8 and S100A9 in AML, we treated secondary H9M1 mice with S100A8 or S100A9 proteins i.p three times per week. Interestingly, injection of S100A8 had no effect on AML latency, but S100A9 treatment resulted in significant delays of leukemia symptoms suggesting an anti-leukemic activity (32 days vs 41 days, p=0.0045). The extent of increased survival induced by S100A9 treatment was similar to standard induction chemotherapy using combination of doxorubicin and cytarabine. Furthermore, S100A9 treatment led to significant cell cycle arrest and an increase of mature cells marker CD11band Gr-1 in bone marrow (76.6%± 1.0% vs 94.8 ± 1.2%, p Since injection of the S100A9 protein and anti-S100A8 antibody had similar effect on AML progression and cellular differentiation, we postulated that cell differentiation is regulated by the balance between S100A9 and S100A8. To test the hypothesis, cells were cultured in vitro in presence of different ratio of S100A9 on S100A8. At high ratio (S100A9>S100A8), the percentage of CD11b+ Gr-1+ was increased compared to the control suggesting that leukemic cells underwent differentiation. Nevertheless, the augmentation of S100A8 level prevented the increases of CD11b+ Gr-1+ mediated by S100A9. To test the ability of S100A9 protein to promote terminal cell differentiation of human leukemia, human cord blood (CB) CD34+ cells were transduced with retrovirus expressing the oncogene MLL-AF9. In vitro, S100A9 induced a 10-fold up-regulation of CD14 expression in MLL-AF9 cells. More importantly, the increase of CD14 was associated with morphological changes typical of terminal differentiation into monocytes and then macrophages. To determine the receptor(s) involved in regulation of cellular differentiation induced by S100A9 in human AML, we followed CD14 expression in presence of anti-TLR neutralizing antibodies. Blockage of TLR4 and TLR2 prevented the differentiation of human leukemic cells mediated by S100A9. Taken together, we show that increasing the S100A9/S100A8 ratio in murine AML, either by anti-S100A8 antibody or recombinant S100A9 protein, prolong the survival of secondary mice in vivo by inducing differentiation on AML cells. We corroborated these data in human MLL-AF9 cells in vitro and show that S100A9 protein induces terminal differentiation through TLR receptors which could represent a new therapeutic target to explore. Disclosures No relevant conflicts of interest to declare.

SAM68 (Src-associated in mitosis 68 kDa) is a member of the signal transduction of activator RNA novel gene family coding for proteins postulated to be involved in signal transduction and activation of RNA. It has been implicated through its phosphorylation status in the control of the transition from the G1 to the S phases during mitosis. However, the implication and role of SAM68 in nonproliferative cells are unknown. The present study was initiated to examine the role of SAM68 in the phagocytic responses of the terminally differentiated human neutrophils. The results obtained show that SAM68 is present in human neutrophils and that it is tyrosine phosphorylated in response to stimulation by monosodium urate crystals or by ligation of CD32. Stimulation of neutrophils by these agonists decreases the association of SAM68 with Sepharose-conjugated poly-U beads. Additionally, the amount of immunoprecipitable SAM68 was modulated differentially after stimulation by monosodium urate crystals or by CD32 engagement indicating that the posttranslational modifications and/or protein associations of SAM68 induced by these two agonists differed. The results of this study provide evidence for an involvement of SAM68 in signal transduction by phagocytic agonists in human neutrophils and indicate that SAM68 may play a role in linking the early events of signal transduction to the posttranscriptional modulation of RNA.

The inflammatory reaction associated with the deposition of monosodium urate (MSU) crystals in synovial spaces is known to be due to interactions with polymorphonuclear neutrophils mediated by presently unidentified surface structures. In this study, we have observed that antibodies directed against CD16 (VIFcRIII) and CD11b (VIM12) selectively and potently inhibit the activation of neutrophils by MSU crystals. The responses affected include the stimulation of tyrosine phosphorylation, activation of the tyrosine kinase syk, tyrosine phosphorylation of the proto-oncogene Cbl, mobilization of calcium, and stimulation of the activity of phospholipase D and of the production of superoxide anions. Tyrosine phosphorylation responses to MSU crystals develop during the Me2SO4-induced differentiation of HL-60 cells in parallel with the surface expression of CD16. These data strongly support the hypothesis that inflammatory microcrystals interact opportunistically with CD16 initially, and that the signal transduction pathways activated thereby depend on CD11b. An examination of the relevance of the hypothesis that an uncontrolled activation of CD16/CD11b may play a role in inflammatory reactions associated with a dysregulation of neutrophil function (other than crystal arthropathies) appears warranted on the basis of the present results.

In a previous gain-of-function screen, we identified 18 nuclear factors that enhance mouse hematopoietic stem cell (HSC) activity in vitro. Of these factors, the majority was believed to augment HSC function intrinsically. In the current study, we investigated the mechanisms of action of the previously identified agonists of HSC activity and tested whether human HSCs are also responsive to these factors. Our results unexpectedly revealed that the majority of the identified factors confer a competitive advantage to mouse HSCs in a non-cell-autonomous manner. Five of these factors, namely FOS, SPI1, KLF10, TFEC, and PRDM16, show robust transcriptional cross-regulation and are often associated with osteoclastogenesis. These findings define at least one novel non-cell-autonomous network in engineered niches. Surprisingly, and in contrast to their important effect on mouse HSCs, all engineered niches failed to significantly enhance the activity of human HSCs. This last finding further supports a lack of conservation in determinants that control HSC self-renewal in mouse versus human cells.

634 Background: Trastuzumab is well tolerated without major side effects except for cardiac toxicity. Although a number of clinical parameters have been associated with trastuzumab-associated cardiac toxicity (TACT), there is some indication that genetic variation of the HER2 gene may play a toxic role in a population of metastatic breast cancer patients. However, this finding needs confirmation and we looked at a population of non-metastatic breast cancer. This study aimed to evaluate the association between cardiac toxicity and HER2 [Ile655Val] polymorphism in non-metastatic breast cancer patients treated with trastuzumab. Methods: The Ile655Val HER2 polymorphism was assessed in 73 women using TaqMan technology. For this study, the genotyping was performed using DNA extracted from normal breast tissue located at more than 1 cm of any other lesions. Charts review was used to collect information on TACT which was defined as any decline in LVEF > 10 % from the baseline to < 55 % or a decline in LVEF > 5 % to < 50 % (lower limit of normal). The Fisher exact test was used to evaluate the association between cardiac toxicity and HER2 polymorphism. Results: No deviation from the Hardy-Weinberg equilibrium has been observed for the allele and genotype frequencies. The distribution of HER2 polymorphism was 3 Val/Val (4%), 18 Ile/Val (25%) and 52 Ile/Ile (71%). In this population, 19% (14/73) developed a cardiac toxicity. We found that 29% (6/21) of Ile/Val or Val/Val carriers compared to 15% (8/52) of Ile/Ile carriers showed TACT, but this association did not reach statistical significance (P = 0.21). Conclusions: HER2 Ile655Val polymorphism may be an efficient marker of TACT considering this tendency with this small cohort of patients. Larger sample is needed to strengthen this conclusion, since this result may influence on prescribing decision for adjuvant chemotherapy and anti-HER2 therapy in HER2 positive patients.

Abstract 1325 New innovative strategies are needed to expand human hematopoietic stem cells (HSCs) for clinical applications. In this perspective, we recently developed an in vitro/in vivo screening strategy, which revealed 18 nuclear factors that enhance HSC activity (Deneault et al., Cell 2009). During these experiments, mouse HSCs were kept for 12 days in mini-cultures that included viral producer cells for each tested factor. Interestingly, 4 of the 18 hits identified in this initial screen, i.e., Fos, Tcfec, Hmgb1 and Sfpi1 operated through non-HSC autonomous (NHA) mechanisms: expanded HSCs were not infected with retrovirus. This suggested that the transfected viral producer cells (feeder cells) produced membrane-bound or soluble molecules that promote expansion of HSCs introduced in these cultures. We now provide evidence that seven additional factors, i.e., Smarcc1, Vps72, Sox4, Klf10, Ski, Prdm16 and Erdr1 significantly enhance HSC activity through NHA mechanisms, hereafter called “NHA factors”. Moreover, we found that Vps72, Fos and Klf10 also promote expansion of human HSCs by NHA mechanisms. Interestingly at least for feeders expressing Klf10, we observed that physical contact between HSCs and these engineered support cells was not necessary, suggesting the presence of secreted molecule(s) in the medium. Expression profiling was next performed using mRNA extracted from feeder cells transduced with each of the NHA factors. Firstly, we shed light on the transcriptional make up and potential convergence of signaling pathways in the engineered feeder cells: our results reveal two independent but interconnected transcriptional regulatory subnetworks. Strikingly, some constituents of the subnetworks, i.e., Sfpi1, Fos, Klf10 and Tcfec (Mitf-related) have previously been shown to play critical roles in the regulation of osteoclasts, which are a myeloid-derived population of cells residing in the HSC bone marrow niche. In addition, all of the NHA factors act in concert to increase Prdm16 expression levels in a range from 2.5- to 54.6-fold. For this reason, Prdm16 clearly holds the position of central hub of the osteoclastic network. However, Tcfec represents the ultimate downstream effector of this pathway as Prdm16 elevates its expression levels by 18.9-fold. In parallel, Prdm16 can also increase the expression of Sfpi1, which in turn can upregulate Tcfec expression up to 874-fold. Moreover, upregulated mRNA targets corresponding to factors that are secreted or associated with the plasma membrane were considered as potential candidate agonists of HSC self-renewal. A high degree of overlap was observed between the sets of proteins produced by feeder cells engineered to overexpress Sfpi1, Fos, Klf10, Tcfec or Ski. These secreted and membrane bound proteins include Agt, Aspn, Ogn, Ptgds, Nckap1l, Rgs16 and Lcn2. Studies are ongoing to characterize the contribution of these newly identified NHA proteins in HSC expansion. Validation of these NHA proteins in human HSC expansion will have a clear potential for translational medicine. Disclosures: No relevant conflicts of interest to declare.

Abstract 2956 Acute leukemias in newborns, children, adults and elderly share the same name, but their genetic, biology and prognosis are significantly different. These differences may be explained by many factors including the nature of the recurrent genetic abnormalities, tolerance to chemotherapy, co-morbidities, etc. One noteworthy potential difference is the cell of origin in which the initial genetic event occurs, which can be a fetal liver, cord blood or adult bone marrow stem/progenitor cell. Genetically engineered mouse models of leukemias and gene expression studies on human acute lymphoblastic leukemias (ALL) suggest that leukemias developing in utero (i.e. in an early ontogenic cell of origin) are biologically distinct from child and adult leukemias. However, direct comparison of human hematopoietic stem/progenitor cells at different stages of ontogeny has not been tested experimentally. Over 70% of infant acute leukemias and 5–10% of adult leukemias exhibit rearrangements of the mixed-lineage leukemia gene (MLL). Therefore, MLL translocations are highly relevant oncogenes to investigate the leukemic potential of the different cells of origin. The MLL-ENL (MLL-MLLT1) fusion gene, from the t(11;19) (q23;p13.3), is one of the most frequent MLL fusion and can occur in infants and adults. It is found in both acute myeloid leukemias (AML) and ALL (both B-cell and T-cell ALL) and we have previously shown that it generates human B-ALL in immunodeficient mice when introduced into lineage depleted human cord blood cells using a retrovirus (Barabé et al, Science 2007). The same experimental design was used to study the cells of origin; CD34+ cells from human fetal liver (FL), cord blood (CB) and adult bone marrow (BM) were infected with either a retrovirus encoding MLL-ENL and an enhanced green fluorescent protein (EGFP) marker gene, or a control retrovirus encoding EGFP only, then injected into sub-lethally irradiated immunodeficient mice. After transduction with MLL-ENL, FL cells generated leukemias in 8/10 mice, CB cells in 6/6 mice and adult BM cells in only 2/7 mice, although four times more cells were transduced and injected. Adult BM cells were very difficult to infect with retroviruses and were clearly not as potent to reconstitute a human graft in immunodeficient mice in comparison to CB cells. All generated leukemias were B-ALL, regardless of the cell of origin. The leukemic phenotype (CD19+, HLA-DR+, CD20-, IgM-, IgG-, CD34 variable) was similar in all three cell types. Mice injected with MLL-ENL-transduced FL and adult BM cells that were not leukemic had a normal human graft but no EGFP cells. The frequency of leukemia-initiating cells (L-IC) was evaluated at 0.2–1% by limiting dilution in secondary mice and no significant differences were observed between the three cell types. Leukemic cells were harvested from the BM of primary mice and cultured in vitro under conditions supportive of both B-lymphoid and myeloid cells. All leukemias were able to grow in vitro and at least one culture derived from each cell type (FL, CB and adult BM) switched to a myeloid phenotype (monocytic morphology with CD33+ and loss of CD19), demonstrating that leukemias derived from all 3 cell types still have myeloid potential. Thus, our results suggest that MLL-ENL can generate leukemia in FL, CB and adult BM CD34+ cells. The lower penetrance of leukemia with adult BM cells is probably due to the difficulty to infect these cells and their limited reconstitution ability. MLL-ENL induces a leukemic program in all three cell types with similar clinical features, cell surface phenotype, frequency of L-IC and conserved bi-lineage potential (B and myeloid). Gene expression profiling and clonality studies are currently performed on leukemias generated from the 3 different cell types. So far, no significant differences between FL, CB and adult BM as cells of origin of MLL leukemias have been identified, suggesting that the nature of the oncogenic hit is probably more important than the ontogenic stage of the cell of origin. Disclosures: No relevant conflicts of interest to declare.